| Abstract: | 研究背景: 由於嚼食檳榔的盛行，使得口腔癌為台灣男性癌症發生率及死亡率的第四位。90%的口腔癌為鱗狀細胞癌，其特性為容易復發及轉移。近年來發現，腫瘤組織中細胞的異質性引發癌幹細胞存在之可能性。本實驗室先前研究發現，癌幹細胞與上皮-間質細胞轉換過程(epithelial-mesenchymal transformation; EMT) 之相互調控作用對於傳統化療及放射性療法具備阻抗性及導致癌細胞具有高度轉移力，故認為癌幹細胞應是導致口腔癌病患病灶復發致死的主因。因此，尋找有效標靶癌幹細胞特性之療法將有助於口腔癌臨床上預防、診斷、及口腔癌新穎輔佐療法之開發。本實驗室初步分析研究結果發現檜木醇能明顯抑制人類口腔癌幹細胞的增生能力，且對正常口腔細胞無明顯細胞毒性。此外，檜木醇亦可以有效抑制活體內口腔癌幹細胞標記ALDH1活性及CD44的表達、降低幹細胞球體自我更新能力、及細胞侵襲力。綜合上述，檜木醇應為潛力標靶口腔癌幹細胞特性之化學治療物質。微核糖核酸(microRNAs, miRNAs)為重要癌症標記，可與其標的基因結合而降解其表現，在癌症中扮演促癌基因或抑癌基因之角色。初步研究利用miRNAs microarray發現檜木醇會誘發miR-513a-5p之表現。基於上述理由，本研究計畫探討檜木醇可藉由調控miR-513a-5p及其標的基因表現成為有潛力抑制癌幹細胞特性及上皮-間質細胞轉換過程之標靶化學治療物質。 研究目標:第一年目標: 評估檜木醇對口腔癌幹細胞建活體外(in vitro)表型及癌幹細胞標記影響。包含(1)利用MTT assay評估檜木醇對於口腔癌幹細胞增生是否具備抑制力及對口腔正常細胞是否具備毒性傷害; (2) 檜木醇對於口腔癌幹細胞標記ALDH1, CD133, CD44, Oct4, Nanog表現量之影響; (3) 利用幹細胞球體試驗評估檜木醇對於口腔癌幹細胞自我更新能力之影響; (4) 檜木醇對於口腔癌幹細胞in vitro腫瘤生成特性(如爬行、侵襲、血管新生能力) 之影響; (5) 檜木醇作用對於口腔癌幹細胞細胞凋亡能力之影響; (6) 探討檜木醇搭配傳統化療藥物(如cisplatin or 5-fluorouracil)協同作用於口腔癌幹細胞，評估檜木醇是否能提昇口腔癌幹細胞之化療敏感性及降低抗藥性標記。(7) 檜木醇作用是否改變口腔癌幹細胞上皮-間質相互轉換特性。第二年目標: 利用口腔癌幹細胞建立活體內模式(in vivo)驗證檜木醇具備潛力為標靶癌幹細胞新穎治療方針。包含(1) 建立口腔癌幹細胞腫瘤生成及癌轉移老鼠模式; (2)餵食檜木醇搭配傳統化學療法評估裸鼠腫瘤大小、轉移能力、及存活時間; (3)利用免疫組織染色法評估檜木醇作用於老鼠之腫瘤後，其癌幹細胞、上皮間質轉換過程、血管新生等標記之改變量。第三年目標: 檜木醇調控miR-513a-5p之於口腔癌幹細胞之功能性及詳細機制之探討。包含(1) 利用real-time RT-PCR探討檜木醇作用於口腔癌幹細胞之miR-513a-5p表現; (2)研究miR-513a-5p過度表現對口腔癌幹細胞特性之變異；(3)探討miR-513a-5p過度表現對口腔癌幹細胞腫瘤化特性之改變；(4)探究抑制miR-513a-5p表現對非口腔癌幹細胞族群之幹細胞特性之改變；(5)鑑定miR-513a-5p於口腔癌幹細胞之標的基因及後續功能性研究；(6)詳細研究miR-513a-5p於口腔癌幹細胞之分子機轉。 重要性: 本研究強調基礎研究與臨床應用接軌的重要性，提供檜木醇調控miR-513a-5p成為對抗口腔癌幹細胞及標靶上皮-間質相互轉換特性的化學預防治療藥劑以利於後續的功能性研究，並提供多方面的臨床生物指標應用於口腔癌臨床診斷及治療，希望能進一步開發成為標靶治療藥物，提供未來個人化醫療之應用。
Background Due to the popularity of areca use, oral cancer (OC) has become a prevalent disease in Taiwan. OC is currently the 4th most common cancer type and leading cancer mortality in male Taiwanese. Almost 90% of OC is classified as oral squamous cell carcinoma (OSCC), which is characterized by high recurrence and early metastasis. Most patients relapse within months after current therapeutic treatments. A subpopulation of cells called cancer stem cells (CSCs) possessing stemness properties was shown to enrich after therapy, resulting in the relapse and metastasis of tumors. Our previous studies have demonstrated that oral cancer derived cancer stem cells (OC-CSCs) presented high tumorigenic, chemo-radioresistant, metastatic properties, and coupled with gain of epithelial-mesenchymal transition (EMT) characteristics. Thus, an effective therapeutic approach targeting these OC-CSCs cells may help to improve current treatment regimens for OC-related malignancies. MicroRNAs (miRNAs)—highly conserved small RNA molecules that regulate gene expression—can act as cancer signatures, and as oncogenes or tumor suppressors depending on its main target genes. In our preliminary results, we showed that hinokitiol inhibited CSCs properties and in vivo tumorigenicity through the miR-513a-5p induction, which resulted in the inhibition of the self-renewal, tumor initiation, and invasion properties of OC-CSCs. Therefore, elevating miR-513a-5p by methods such as hinokitiol treatment appears to be a promising therapeutic modality to target OC-CSCs. Experimental method and specific aims In first year, we will evaluate the role of hinokitiol in the modulation of the CSCs properties and markers of OC-CSCs in vitro, involving (1) Evaluation of hinokitiol on toxicity of normal oral cells and proliferation rate of OC-CSCs; (2) Examination of hinokitiol on targeting CSCs markers including ALDH1 activity, CD133, CD44, Oct4, Nanog expression in OC-CSCs; (3) Assessment of hinokitiol on self-renewal property of OC-CSCs; (4) Determination of hinokitiol on in vitro tumorigenic properties in OC-CSCs; (5) Evaluation of hinokitiol on apoptotic ability of OC-CSCs; (6) Examination of hinokitiol on chemosensitization of OC-CSCs; (7) Analysis of the hinokitiol-mediated EMT properties in OC-CSCs. In the second year study, we will establish animal models to investigate the effect of hinokitiol on anti-CSCs ability and chemosensitizing efficacy involving (1) Establish of the OC-CSCs-derived tumorigenic and metastatic animal models; (2) Validation of anti-tumor and survival rate in nude mice with xenografts of OC-CSCs treated with chemotherapeutic agents by oral gavage alone or combined with chemotherapy in developed severe tumors; (3) Evaluation of hinokitiol in vivo anti-CSCs ability with ex vivo biopsies and immunohistochemistry of CSCs, EMT, and angiogenic markers in xenografts-derived tumor sections by hinokitiol treatment combined with chemotherapy. In the third year study, we will clarify the functional involvement and molecular mechanisms of hinokitiol-mediated miR-513a-5p on OC-CSCs properties, involving (1) Exploration of the specifically expressed miR-513a-5p levels in hinokitiol-treated OC-CSCs by real-time RT-PCR analysis; (2) Investigation of the effect on stemness properties of miR-513a-5p overexpressed OC-CSCs; (3) Determination of the effect of overexpression of miR-513a-5p on in vitro and in vivo tumorigenic properties in OC-CSCs. (4) Examination of the effect on stemness properties of non-oral CSCs with down-regulation of miR-513a-5p; (5) Identification of the target genes controlled by miR-513a-5p; (6) Elucidation of the miR-513a-5p regulated molecular mechanisms of the stemness and tumorigenicity of OC-CSCs in detail. Significance In three years of the integration of basic and clinical translational research study, we expect to identify hinokitiol as anti-CSCs chemotherapeutic agents for a potential chemo-adjuvant therapy against OC, and will be developed as target therapy drugs for personal therapy. |
